Polynucleotide hybridization assays are used as research tools for the detection and identification of unique or specific polynucleotide sequences in samples of complete, fragmented, or mixed nucleic acid. Various hybridization diagnostic techniques have been developed.
Southern, J. Mol. Biol., 98:503 (1975), discloses a polynucleotide hybridization technique employing radiolabeled nucleic acid probes. This procedure permits autoradiographic detection of probe/analyte hybrids and identification of the polynucleotide sequence of the analyte. However, the Southern procedure, as well as the other diagnostic procedures employing radiolabeled nucleic acid probes, are very complex, time consuming, and have the additional problems and expenses generally associated with radioactive materials such as personnel monitoring and disposal. Thus, such assays have remained a tool of basic research and are not generally employed in applied or commercial areas such as clinical diagnosis.
Ward et al., European Patent Application No. 82301804.9, Publication No. 0063879, published June 4, 1982, discloses compositions useful as probes in biomedical research and recombinant DNA technology, wherein said probes comprise purine, 7-deazapurine or pyrimidine covalently coupled to a moiety capable of forming a detectable complex with a polypeptide, said moiety being coupled to purine bases at the 8-position, to deazapurine bases at the 7-position, or to pyrimidine bases at the 5-position to form a modified nucleotide. The resulting modified nucleotides are incorporated into DNA by nick-translation techniques.
Ranki, European Patent Application No. 82306489.5, Publication No. 0079139, published May 18, 1983, discloses a technique for the sandwich hybridization of nucleic acids, said technique comprising contacting single-stranded nucleic acid from a microorganism with a pair of different nucleic acid reagents, both reagents of the pair being single-stranded and complementary with the microorganism-derived nucleic acid and one of the pair being a nucleic acid fragment attached to a solid carrier, such as a nitrocellulose filter, while the other is a nucleic acid fragment labeled with a radioactive marker, whereby a labeled hybrid is formed attached to the solid carrier, for the identification of a microorganism or group of microorganisms present in a sample. The correctness of the identification is tested by detection of the extent of formation of a labeled hybrid attached to the solid carrier.
Tchen et al., PCT Application No. PCT/FR82/00220, published July 7, 1983, disclose nucleic acid probe compositions which have been chemically modified by the covalent attachment of at least one N-2-acetylaminofluorene group to one of the bases of the nucleic acid. After hybridization with the target homologous nucleic acid sequence, such hybridization may be detected by the use of enzyme-labeled antibodies.
Kourilsky et al., PCT Application No. PCT/FR82/00223 published July 7, 1983, disclose DNA molecules modified by covalent attachment of an oligomer of modified ribonucleotides, or a single modified ribonucleotide, which provides a means for coupling a chemical capable of recognition by another molecule or product.
Co-pending and commonly assigned U.S. application Ser. No. 574,630, discloses polynucleotide probe compositions which contain a polypeptide moiety capable of enzymatically activating a zymogen to initiate a detectable enzymatic reaction cascade.
Falkow et al., U.S Pat. No. 4,358,535 issued Nov. 9, 1982, disclose a method for detecting the presence of a pathogen in a clinical sample by depositing and fixing said sample on an inert support and hybridizing the genetic material of the target pathogen to a labeled-nucleic acid probe. The label may be a radioisotope, a ligand, a fluorescer, a chemiluminescer, an enzyme, or an antibody.
Kourilsky et.al., U.K. patent application No. 7913031, Publication No. 2019408A published Oct., 31, 1979, disclose a method for detecting the possible presence of a DNA fragment in a sample comprising the hybridization of the sought fragment with an RNA probe which is coupled to an enzyme either prior to or subsequent to the hybridization reaction. The possible presence of the target nucleic acid sequence is revealable by the action of the enzyme-labeled hybridization product on a chromogen substrate.
Heller et al., European patent application No. 82303701.5, Publication No. 0070687, published Jan. 26, 1983, disclose a heterogeneous hybridization diagnostic method which uses luminescer-labeled, single-stranded polynucleotide reagents for hybridizing with immobilized sample single-stranded polynucleotides. After separation of the unhybridized reagent, the sample is exposed to light. Any subsequent light emission is related to the amount of target polynucleotide in the sample. The label may be any of the well known luminescent systems.
Heller et al., European patent application No. 82303699.1, Publication No. 0070685, published July 14, 1982, disclose a homogeneous light-emitting hybridization assay wherein luminescer-labeled first and second single-stranded reagent segments are hybridized with a complementary target single-stranded polynucleotide from a physiological sample such that nonradioactive energy transfer occurs between the labels of the two reagent segments. At least one of the labels is of the absorber/emitter type such that energy in the form of a photon absorbed from the other light label is re-emitted as a different wavelength. Such secondary emissions can only occur if hybridization has taken place. This system, however, suffers the disadvantage of requiring two luminescer-labeled probes of absorber/emitter character distinct enough to be able to differentiate one from the other by photometric means. Such differentiation may be difficult in complex physiological samples.
The enzyme catalyzed conversion of apoluminescer to luminescer is known. Brandt et al., Anal. Biochem., II, 6 to 9 (1965); and Keston et al. Anal. Biochem., II, 1 to 5 (1965); disclose the conversion of the nonfluorescent apofluorophore diacetyl 2',7'-dichlorofluorescen to a fluorescent compound by hydrogen peroxide and peroxidase for the fluorometric analysis of ultramicro quantities of hydrogen peroxide. Guilbault et al. Anal. Chem., 40 (8), 1256 to 1263 (1968); Brunvoll, Acta Chem. Scand., 21 (3), 820 to 821 (1967): and Guilbault et al., Anal. Chem., 39 (2), 271 (1967), disclose the conversion of the nonfluorescent apofluorophores homovanillic acid, p-hydoxyphenylacetic acid, tyrosine or tyramine to a fluorescent compound by hydrogen peroxide and peroxidase for the fluorometric analysis of ultramicro quantities of hydrogen peroxide. Cathcart, Anal. Biochem., 134, 111 to 116 (1983), discloses the conversion of nonfluorescent apofluorophores to fluorescent compounds by hydrogen peroxide and hematin for the fluorometric detection of picomole levels of hydrogen peroxide. The Cathcart reference also discloses that alternate peroxides could be employed in place of hydrogen peroxide as the substrate for peroxidase or hematin. None of the above references disclose or suggest the use of apofluorophores as labels for polynucleotide probes.
There is a need in the area of clinical diagnostics for a nonradiometric homogeneous assay which is fast simple to carry out, highly specific and highly sensitive. The assay of the present invention fulfills this need.